ORIGINAL ARTICLE
Differential effect of short-term etidronate treatment on
three cancellous bone sites in orchidectomized adult rats
Jun Iwamoto, Tsuyoshi Takeda and Shoichi Ichimura1
Department of Sports Medicine, Keio University School of Medicine, 1Department of Orthopaedic Surgery
Kyorin University School of medicine, Tokyo, Japan(Received for publication on May 28, 2003)

Abstract. The aim of the present study was to analyze the effects of short-term treatment with theantiresorptive agent, etidronate, on orchidectomized adult rats, via comparison of three cancellous
bone sites, the lumbar vertebral body (LVB), proximal tibial metaphysis (PTM), and distal tibial
metaphysis (DTM). Thirty-ﬁve male Wistar rats, aged 10 months, were randomly divided into fourgroups: baseline control (BLC, n F 10), age-matched sham-operated control (AMC, n F 9), orchidectomy (ORX, n F 9), and ORXBetidronate treatment (n F 7). Etidronate treatment (10 mg/kg, daily
subcutaneous injection) was initiated 2 weeks after surgery and was continued for 2 weeks. Four weeks
after surgery, the 5th LVB, PTM, and DTM were processed for histomorphometric analysis of cancellous bone (secondary spongiosa). ORX resulted in a decrease in body weight. No signiﬁcant difference in cancellous bone volume (BV/TV) was found between the BLC and AMC groups at any skeletal site. The cancellous BV/TV loss was attributable to increased eroded surface (ES/BS) with nosigniﬁcant alteration in the mineral apposition rate (MAR), at all skeletal sites and etidronate treatment in ORX rats signiﬁcantly decreased ES/BS to a level not signiﬁcantly different from that in the
AMC group, resulting in complete prevention of ORX-induced cancellous BV/TV loss. The MAR was
markedly decreased in the PTM and LVB, but maintained in the DTM by etidronate treatment. The
present study showed that etidronate treatment could completely prevent ORX-induced cancellous
bone loss regardless skeletal sites by suppressing bone resorption. In particular, suppression of boneformation in terms of osteoblastic activity by etidronate treatment was not evident only in the DTM.
(Keio J Med 53 (1): 12–17, March 2004)
Key words: orchidectomy, etidronate, bone histomorphometry, distal tibial metaphysis (DTM), adultrats

To date, it has been reported that ovariectomy in 3month-old rats does not induce cancellous bone loss inthe distal tibial metaphysis (DTM) with no alteration inbone formation endpoints, and treating ovariectomized
rats with prostaglandin-E2 prevents age-related bonechanges, adds extra bone, and improves the microanatomical structure by stimulating bone formation
without altering bone resorption.9 It has also been reported that prostaglandin-E2 treatment induces morebone formation in the DTM than in the PTM in adultrats.10 The DTM has a fully closed growth plate in adultrats because the growth plate closes at 3 months.11

Introduction
The orchidectomized rat model has been widely
accepted for studying the prevention of androgendeﬁcient cancellous bone loss.1–5 Testosterone deﬁciency induced by orchidectomy (ORX) inhibits cancellous bone gain in rapidly growing male rats, andinduces cancellous osteopenia in adult rats as a result of
increased bone turnover.6–8 Most of the studies havefocused on the proximal tibial metaphysis (PTM), distal
femoral metaphysis, and lumbar vertebral body (LVB),
which are sites with a nonfused growth plate.

Reprint requests to: Dr. Jun Iwamoto, Department of Sports Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan, e-mail: jiwamoto@sc.itc.keio.ac.jp
12Keio J Med 2004; 53 (1): 12–17

Thus, the cancellous bone response to treatment
with an anabolic agent, prostaglandin-E2 in the DTM
has been considered greater than that in the PTM in
adult rats. However, very few studies have reported thealteration of cancellous bone in the DTM of orchidectomized adult rats, and the comparative response ofcancellous bone in the DTM and PTM to antiresorptive
agents has rarely been reported, although it was reported that etidronate treatment prevented loss of bonemineral density in the proximal tibia, distal femur, and
LVB in ORX rats.12 The aim of the present study was
to compare the response of cancellous bone to ORX
and short-term etidronate treatment for ORX-induced
cancellous osteopenia among the three skeletal sites,
the LVB, PTM, and DTM in adult rats.
Materials and Methods
Treatment of animals
Forty 4-week-old male Wistar rats were purchasedfrom SLC Japan Inc. (Tokyo, Japan) and acclimatizedfor 9 months under standard laboratory conditions at
a room temperature of 23 G 2 C and a humidity of
55 G 5%. The rats were allowed free access to tap
water and commercial standard rodent chow (CE-2:
Clea Japan Inc., Tokyo, Japan) containing 1.25% calcium, 1.06% phosphorus and 2.0 IU/g vitamin D3 .
Rats aged 10 months were used. Ten rats served asbaseline control (BLC). The remaining 30 rats underwent ORX or sham-operation under ether anesthesia,
and were randomly divided into three groups of 10 rats
each: age-matched sham-operated control (AMC),
ORX and ORX þ etidronate treatment (E). Body
weight was measured every day. Bisphosphonate etidronate was synthesized at Sumitomo Pharmaceuticals(Osaka, Japan). Etidronate treatment (10 mg/kg bodyweight, daily subcutaneous injection) was initiated 2
weeks after surgery when the surgical wound was completely healed, and was continued for 2 weeks. The
dose of etidronate was determined based on the resultsof a previous study.13 During the experiment, one rat ineach of the AMC and ORX groups and three rats in the
ORX þ E group were removed from the study because
they spontaneously died within several days postsurgery probably as a complication of surgery. Animal
breeding was performed at Sumitomo Pharmaceuticals
Research Center, Osaka, Japan. These animal studies
were carried out in accordance with Sumitomo Pharmaceuticals’ ethical guidelines for animal care.
Preparation of specimens
Rats in the BLC group were killed by exanguination
under ether anesthesia at the beginning of the experi-

13

ment. Rats in the AMC, ORX, and ORX þ E groups
were labeled by subcutaneous injection of 8 mg/kg calcein and 20 mg/kg tetracycline at 14 days and 7 days
before killing, respectively. They were killed by exsanguination under ether anesthesia at 4 weeks after surgery. The left tibia and 5 th LVB were removed andstored in 70% ethanol at 4 C. The tibia was cut using a
diamond band saw and divided into three parts: theproximal tibia, middle tibia, and distal tibia. The LVB
was also isolated using a diamond band saw. The PTM,
DTM, and LVB were ﬁxed in 70% ethanol and stained
according to the method of Villanueva.14 After dehydration with ethanol and acetone, the samples wereembedded in methylmethacrylate (Wako Pure Chemical, Japan). Frontal sections of the PTM and sagittal
sections of the DTM and LVB were cut at 5 mm thickness using a microtome (Supercut 2050, Reichert-Jung,
Heidelberg, Germany) and prepared for histomorphometric analysis of cancellous bone.
Bone histomorphometry
The image of the specimen, observed under a ﬂuorescence microscope and recorded with a video camera(DK-3000, Hitachi, Japan), was processed using a plotter (Cosmozone 1SA, Nikon, Japan). The following
primary parameters for cancellous bone were measured: total tissue volume (TV, mm 2 ), bone volume(BV, mm 2 ), bone surface (BS, mm), eroded surface (ES,mm), single-labeled surface (sLS, mm), double-labeledsurface (dLS, mm), and interlabel width (IrLWi, mm).
From these primary parameters, the following parameters were calculated according to the standard nomenclature described by Parﬁtt, et al.15: bone volume (BV/
TV, %), eroded surface (ES/BS, %), mineralizing surface (MS/BS, %), mineral apposition rate (MAR, mm/day), bone formation rate/BS (BFR/BS, mm 3 /mm 2 /day),and BFR/BV (%/year). In the present study, the regionof cancellous bone measured was 1–3 mm distal to thegrowth plate for the PTM, 0–2 mm proximal to theclosed growth plate for the DTM, and 1–3 mm cranial
to the caudal growth plate for the LVB. All of thoseareas corresponded to the secondary spongiosa.
Statistical analysis
All data in the tables and ﬁgures are presented asmean G standard deviation (SD). The changes in bodyweight with time in the AMC group were evaluated by
one-way analysis of variance (ANOVA) with repeatedmeasurements. The changes in body weight with time
were compared between the AMC and other groups
using two-way ANOVA with repeated measurements.
Multiple comparisons of data such as body weight ateach time point and bone histomorphometric parame-

14
Table 1

Iwamoto J, et al: Effect of etidronate on cancellous bone
Changes in Body Weight

BLC
AMC
ORX
ORX þ EnBaseline

Day 7

Day 14

Day 21

Day 28

Two-way ANOVA

10
9
9
7

468 G 20469 G 21470 G 20469 G 17458 G 24
434 G 22a
443 G 9460 G 24
443 G 18445 G 11468 G 21455 G 13460 G 13469 G 24456 G 12458 G 11

P < 0.05
P < 0.05

a P < 0.05 vs AMC group.

Fig. 1 Cancellous BV/TV. Data are presented as means and SD. * P < 0.001, ** P < 0.05 vs BLC group. a: P < 0.001, b: P < 0.01, c: P < 0.05
vs AMC group. # P < 0.01 vs ORX group. BV/TV: bone volume/tissue volume.

ters among the groups were performed by ANOVA
with Fisher’s protected least signiﬁcant difference(PLSD) test. P < 0.05 was considered statistically signiﬁcant. All statistical analyses were performed using
the Stat View J-5.0 program on a Macintosh computer.
Results
Body weight
Table 1 shows the changes in body weight. Baseline
body weight did not differ signiﬁcantly among thegroups. The longitudinal changes in body weight in the
AMC group were not signiﬁcant (one-way ANOVA).
Body weight in the ORX and ORX þ E groups signiﬁcantly decreased longitudinally compared with the
AMC group (both P < 0.05, two-way ANOVA).
Bone histomorphometry
Figure 1 and Tables 2, 3, and 4 show the structuralvariables and the formative and resorptive variables ofcancellous bone in the PTM, DTM, and 5th LVB. No
signiﬁcant difference in cancellous BV/TV was foundbetween the BLC and AMC groups at any skeletal site.

Bone turnover, as indicated by BFR/BV in the AMC
group, was lower in the DTM than in the PTM andLVB and lower in the LVB than in the PTM. However,
the magnitude of ORX-induced cancellous BV/TV loss
greater in the PTM and DTM than in the LVB andgreater in the DTM than in the PTM. The cancellous
BV/TV loss was attributable to increased ES/BS withincreased MS/BS and no signiﬁcant alteration in MAR.
The ORX-induced increase in ES/BS was greater in the
PTM and DTM than in the LVB and greater in the
DTM than in the PTM.
Etidronate treatment in ORX rats signiﬁcantly decreased ES/BS to a level not signiﬁcantly different fromthat in the AMC group at all skeletal sites, resulting incomplete prevention of ORX-induced cancellous BV/
TV loss. In the LVB and PTM, etidronate treatment
markedly decreased MAR and MS/BS and subsequently BFR/BS and BFR/BV. In the DTM, however,
although MS/BS was signiﬁcantly decreased by etidronate treatment, MAR was maintained.
Discussion
ORX has been reported to induce high turnovercancellous osteopenia in adult rats. In particular,Keio J Med 2004; 53 (1): 12–17
Table 215

Bone Histomorphometry of PTM

Parameters
ES/BS (%)
MS/BS (%)
MAR (mm/d)
BFR/BS (mm 3 /mm 2 /d)
BFR/BV (%/yr)

AMC

ORX

ORX þ E

3.55 G 1.18
5.54 G 3.00
0.75 G 0.08
0.043 G 0.026
37.5 G 24.5

7.02 G 2.40a
15.83 G 5.10a
0.76 G 0.09
0.121 G 0.040a
102.9 G 34.0a

3.28 G 0.71#
0.48 G 0.18b,#0.00 G 0.00a,#0.000 G 0.000b,#
0.0 G 0.0a,#

Data are means G SD. a P < 0.001, b P < 0.01 vs AMC group, # P < 0.001 vs ORX group. PTM: proximal tibial metaphysis, ES: eroded surface,
BS: bone surface, MS: mineralizing surface, MAR: mineral apposition rate, BFR: bone formation rate, BV: bone volume.

Table 3

Bone Histomorphometry of DTM

Parameters
ES/BS (%)
MS/BS (%)
MAR (mm/d)
BFR/BS (mm 3 /mm 2 /d)
BFR/BV (%/yr)

AMC

ORX

ORX þ E

2.61 G 0.92
4.94 G 2.33
0.79 G 0.11
0.040 G 0.025
8.47 G 4.12

7.99 G 2.23a
7.28 G 3.15b
0.79 G 0.08
0.057 G 0.026b
18.1 G 13.5b

1.33 G 0.56#
2.47 G 0.96b,#
0.79 G 0.10
0.020 G 0.010b,#7.61 G 5.45##

Data are means G SD. a P < 0.001, b P < 0.05 vs AMC group, # P < 0.001, ## P < 0.01 vs ORX group. DTM: distal tibial metaphysis, ES: eroded
surface, BS: bone surface, MS: mineralizing surface, MAR: mineral apposition rate, BFR: bone formation rate, BV: bone volume.

Table 4

Bone Histomorphometry of LVB

Parameters
ES/BS (%)
MS/BS (%)
MAR (mm/d)
BFR/BS (mm 3 /mm 2 /d)
BFR/BV (%/yr)

AMC

ORX

ORX þ E

2.60 G 0.89
5.08 G 1.38
0.70 G 0.17
0.037 G 0.019
25.5 G 13.1

3.86 G 0.80c
14.66 G 4.58a
0.73 G 0.07
0.108 G 0.036a
80.8 G 25.2a

2.02 G 1.01##
1.25 G 1.15c,#
0.00 G 0.00a,#0.000 G 0.000b,#
0.0 G 0.0c,#

Data are means G SD. a P < 0.001, b P < 0.01, c P < 0.05 vs AMC group, # P < 0.001, ## P < 0.01 vs ORX group. LVB: lumbar vertebral body,
ES: eroded surface, BS: bone surface, MS: mineralizing surface, MAR: mineral apposition rate, BFR: bone formation rate, BV: bone volume.

Erben, et al.6 clearly demonstrated that ORX-induced
osteopenia in the PTM and LVB of adult rats was
associated with increased osteoclast number, osteoblast
surface, bone formation rate, and activation frequency.
The effect of ORX on cancellous bone in the DTM has
rarely been reported. In the present study, ORX
induced cancellous BV/TV loss similarly in the PTM,
DTM, and LVB, which was associated with increased
eroded surface and bone formation rate with increasedbone turnover (ES/BS and BFR) with increased osteoblastic recruitment (MS/BS) and no signiﬁcant alteration in osteoblastic activity (MAR).
Bone turnover was lower in the DTM than in the
PTM and LVB and lower in the LVB than in the PTM
according to the results of bone histomorphometricanalysis in the AMC group. The ORX-induced increasein ES/BS was greater in the PTM and DTM than in the

LVB and greater in the DTM than in the PTM. We
surmise that ORX-induced body weight loss in terms ofdecreased body weight loading and/or the amount ofprogenitor cells inside the cortex participating in forming bone in the marrow cavity may somewhat inﬂuence
the differential response of cancellous bone to ORX
among the PTM, DTM, and LVB.
The tibia differs from the lumbar vertebra in the rat
not only in the ratio of cortical to cancellous bone, butalso in its position in the body where there is more bodyweight loading on the tibia relative to the lumbar vertebra. Furthermore, the DTM is a bony region closest
to the point of weight loading (the most distal aspectof the tibia), and contains a thicker metaphyseal cortex than the PTM. The DTM, the distal aspect of
the weight-bearing limbs is in closer contact with theground and is thus loaded more directly. Therefore, the16

DTM receives more body weight loading under usual
activity, and furthermore contains much more progenitor cells than the PTM.16 Body weight change affects
mechanical stress loaded on the tibia much more thanthat loaded on the lumbar vertebra under usual activity
in rats, and also affects mechanical stress loaded on the
DTM more than that loaded on the PTM. The larger
number of progenitor cells in the DTM can bring about
greater bone formation than in the PTM.
In fact, the effect of mechanical stress on bone issuggested to be site-speciﬁc. The effect of treadmill
exercise on cancellous bone mass in rats is greater inthe tibia than in the lumbar vertebrae and greater in the
DTM than in the PTM.17 Conversely, bone loss in theweight-bearing limbs due to disuse or bed rest is greatest in bony regions closest to the point of loading (i.e.,the most distal aspects).18,19 The anabolic response ofcancellous bone to exercise is largest at the most distal
aspects of weight-bearing limbs, and the catabolic response of bone to disuse is similarly most severe. Disuse
tends to suppress bone formation and increase bone
resorption.20,21 In the present study, despite lower
turnover, cancellous BV/TV loss was greater in the
DTM than in the PTM, probably because ORXinduced body weight loss affected the DTM more than
the PTM, resulting in more accelerated bone resorption(indicated by percent increases in ES/BS). Bone formation was not suppressed by decreased body weightloading in the DTM, probably because of the effect of alarger number of progenitor cells. On the other hand,
the less marked cancellous BV/TV loss in the LVB as
compared with the PTM may be attributable to lower
turnover and smaller body weight change effect.
Another possible explanation for the differential response of cancellous bone to ORX between the PTM
and the DTM concerns the pressurization of the
medullary cavity of the tibia.19 Bergula, et al.22 demonstrated that inhibition of femoral bone apposition
caused by disuse could be mitigated by femoral vein
ligation in rats, which signiﬁcantly increased the intramedullary pressure in the femur. Thus, changes inpressure of the medullary cavity may possibly affectbone mass. The distal aspects of the hindlimbs are thefurthest points from the heart and are therefore subject
to higher interstitial ﬂuid pressure. Because androgen
has an anabolic effect on muscle, ORX might possibly
reduce muscle volume, resulting in a reduction of blood
ﬂow and subsequent pressure of the medullary cavity ofthe tibia in the present study. This change in pressure of
the medullary cavity of the tibia might partly affect theDTM more than the PTM.
Bisphosphonates may inhibit osteoclast-mediatedbone resorption, and loss of osteoclast function andapoptosis is probably the consequence of loss of function of one or more these important signaling proteins.

Iwamoto J, et al: Effect of etidronate on cancellous bone

In particular, etidronate can be metabolically incorporated into nonhydrolyzable analogs of ATP, and intracellular accumulation of these metabolites is likely toinhibit osteoclast function. In the present study, shortterm treatment with etidronate did prevent cancellous
BV/TV loss in the PTM, DTM, and LVB of ORX rats.
The primary effect of etidronate treatment in ORX rats
was a reduction of bone resorption. However, in the
LVB and PTM, etidronate treatment markedly decreased MAR, MS/ BS, and subsequently BFR/BS andBFR/BV. In the DTM, on the other hand, although
MS/BS was signiﬁcantly decreased by etidronate treatment, MAR was maintained. This result in the DTM
may be attributable to the effect of a larger number ofprogenitor cells in the DTM. The effect of the amountof progenitor cells may play a role in the differential
response of cancellous bone to etidronate treatment in
the PTM and DTM.
Major limitations of the present study are associatedwith use of a single dose of etidronate and shortduration of treatment. First, although the dose of etidronate was determined based on the results of a previous
study,13 this dose might however, be so high in our
rat model that a mineralization defect (indicated by amarked reduction in mineral apposition rate) was foundin the PTM and LVB, even though cancellous BV/TV
loss was completely prevented. Thus, a minimum effective dose should have been identiﬁed in our rat model,
and it should have been determined whether use of thisdose of etidronate would not cause any mineralization
defect. Second, although ORX resulted in signiﬁcant
cancellous BV/TV loss 4 weeks postsurgery, long-term
treatment with etidronate might be more useful, if thisstudy is applied as a preclinical study. Further study oflong-term duration of treatment is needed to conﬁrm
the efﬁcacy of etidronate for ORX-induced cancellous
osteopenia.
In conclusion, the results of the present study suggestthat the response of cancellous bone mass to ORX
appears to differ among three skeletal sites. Etidronate
treatment could completely prevent ORX-induced cancellous bone loss regardless skeletal sites by suppressing bone resorption. However, suppression of boneformation in terms of osteoblastic activity by etidronate
treatment was not evident only in the DTM.
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